Cellulase producing microorganism ATCC 55702

ABSTRACT

Bacteria which produce large amounts of cellulase--containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualifies for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

This invention was made with Government support under contractDE-AC05-84OR21400 awarded by the U.S. Department of Energy to LockheedMartin Energy Systems, Inc. and the Government has certain rights inthis invention.

This application is a division of application Ser. No. 08/528,178 filedSep. 14, 1995, pending.

FIELD OF THE INVENTION

The present invention relates generally to the conversion of cellulosicmaterials into fuels, sugars, and specialty chemicals, and moreparticularly to bacteria which produce cellulase and other enzymes undervarious environmental conditions.

BACKGROUND OF THE INVENTION

Space in industrial and domestic land fills is becoming increasinglyscarce. Therefore, there is need to conserve the space in the existingland fills by reducing the volume of materials via recycling,incineration or other methods. Waste disposable cellulosic materials(i.e., newsprint, diapers, packaging, containers, etc.) are majorcontributors of the total volume placed in land fill sites. Cellulosicmaterials are generally defined as those materials which containcellulose. Cellulose is generally defined as a polymer of β-D-glucoseunits. Concern over the environmental consequences of depositing thesematerials has led some environmentally conscious consumers to refrainfrom purchasing cellulosic disposable items. Major international foodsuppliers have recently replaced styrofoam packaging materials with moreenvironmentally conscious cellulosic packaging materials. However, asignificant volume of cellulosic based materials will still need to bedeposited in land fills. Therefore, new methods to enhance thebiodegradation of cellulosic materials are required in order toalleviate this problem. New strains of bacteria with geneticallyenhanced capabilities need to be produced to make these processescommercially feasible.

Additionally, with the increasing dependance on unreliable fossil fuelsources, biomass conversion of waste cellulosic products intoalternative fuels is also an area of intense interest. Waste cellulose,coal, or other cellulosic materials can be converted into products thatcan be used to produce alternative fuels, such as ethanol.

Cellulosic waste streams and fossil fuels often contain hazardousmaterials besides the cellulosic products. To degrade these materials,expansion of the metabolic capabilities of cellulose degrading organismsare required. Efficient modification of these bacteria will probablyrequire recombinant DNA methods. Stable cloning hosts withcharacteristics desirable for rapid expression and selection ofrecombinants are required (i.e., sensitivity to common antibiotics).

Cellulosic waste streams, paper production processes, and fossil fuelsoften produce extremely harsh environmental conditions. Enhancedorganism with tolerances for extreme environments and relatively lowtemperature growth are advantageous for bioconversion of cellulosicmaterials. Conditions for forming bioreactor solid matrices are oftenvery harsh (i.e., very basic pH values). Organisms with enhancedcellulase producing capabilities that can tolerate alkaline conditionsare advantageous for use in bioreactors for converting cellulose wasteor fossil fuels.

Most efforts on cellulose degrading microorganisms has centered on thecellulose degrading fungi (i.e., Trichoderma reesei). However,filamentous fungi are more difficult to grow in a fermentor than amaterial cellulose degrader. Fungi are more difficult than bacteria togenetically engineer (utilizing recombinant DNA methods) to enhancecellulase production or to modify metabolic capabilities.

Most efforts on bacterial cellulases have been concentrated on theanaerobic cellulose degrading bacteria. However, airless fermentationsare more difficult and expensive than those performed with aerobic (airutilizing or air tolerant) bacteria. Many bacteria and fungi producecellulases that: (1) are not efficiently excreted into the surroundinggrowth medium, or (2) cannot efficiently degrade solid cellulosicmaterials. Chemical modification of cellulosic waste is often requiredto make many solid cellulosic materials available for biodegradation.Thermophilic (requiring high temperatures) cellulose degraders are alsoof intense scrutiny for use as producers of alternative fuels.

To make bioreactor conversion of waste commercially practical, newgenetically enhanced organisms are also required. Additionally, toeffectively utilize recombinant DNA methods (genetic engineering) tomodify these organism, acceptable cellulase producing cloning hosts arealso required.

Therefore, there is a need for bacteria which produce large amounts ofcellulase and other enzymes under various environmental conditions.

OBJECTS OF THE INVENTION

Accordingly, it is an object of the present invention to provide a newand improved bacterium with increased enzyme producing ability.

It is another object of the present invention to provide a new andimproved bacterium with increased cellulase producing ability.

It is another object of the print invention to provide a new andimproved bacterial cloning host with increased cellulase producingability.

It is another object of the present invention to provide a new andimproved bacterium with increased enzyme producing ability over a widepH range.

It is another object of the present invention to provide a new andimproved bacterium with increased cellulase producing ability over awide pH range.

It another object of the present invention to provide a new and improvedbacterial cloning host with increased cellulase producing ability over awide pH range.

It is another object of the present invention to provide a new andimproved backfire with increased enzyme producing ability over a widetemperature range.

It a another object of the present invention to provide a new andimproved bacterium increased cellulase producing ability over a widetemperature range.

It is another object of the present invention to provide a new andimproved bacterial cloning host with increased cellulase producingability over a wide temperature range.

It is another object of the present invention to provide a new andimproved bacterium with increased enzyme producing ability over a widesalt range.

It is another object of the present invention to provide a new andimproved bacterium with increased cellulase producing ability over awide salt range.

It is another object of the presto invention to provide a new andimproved bacterial cloning host with increased cellulase producingability over a wide salt range.

It is another object of the present invention to provide a new andimproved bacterium with increased enzyme producing ability and theability to tolerate chemical contaminants.

It is another object of the present invention to provide a new andimproved bacterium with increased cellulase producing ability and theability to tolerate chemical contaminants.

It is another object of the present invention to provide a new andimproved bacterial cloning host with increased cellulase producingability and the ability to tolerate chemical contaminants.

It is another object of the present invention to provide a new andimproved detergent.

It is another object of the present invention to provide a new andimproved method of converting cellulosic materials into fuels.

It is another object of the present invention to provide a new andimproved method of converting cellulosic materials into sugars.

Further and other objects of the present invention will become apparentfrom the description contained herein.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, the foregoingand other objects are achieved by:

a microorganism consisting of ATCC 55703, or a mutant strain thereofpossessing substantially all of the identifying characteristics of themicroorganism.

In accordance with another aspect of the present invention, theforegoing and other objects are a microorganism consisting of ATCC55702, or a mutant strain thereof possessing substantially all of theidentifying characteristics of the microorganism.

In accordance with another aspect of the present invention, theforegoing and other objects are achieved by:

a cellulase produced by a microorganism consisting of ATCC 55703, or amutant strain thereof possessing substantially all of the identifyingcharacteristics of the microorganism.

In accordance with another aspect of the present invention, theforegoing and other objects are achieved by:

a cellulase produced by a microorganism consisting of ATCC 55702, or amutant strain thereof possessing substantially all of the identifyingcharacteristics of the microorganism.

In accordance with another aspect of the present invention, theforegoing and other objects are achieved by:

a method for producing a cellulase, which comprises culturing acellulase-producing microorganism in a medium, the microorganismconsisting of ATCC 55703, or a mutant strain thereof possessingsubstantially all of the identifying characteristics of themicroorganism.

In accordance with another aspect of the present invention, theforegoing and other objects are achieved by:

a method for producing a cellulase, which comprises culturing acellulase-producing microorganism in a medium, the microorganismconsisting of ATCC 55702, or a mutant strain thereof possessingsubstantially all of the identifying characteristics of themicroorganism.

In accordance with another aspect of the present invention, theforegoing and other objects are achieved by:

a composition comprising:

a cellulase produced by a microorganism consisting of ATCC 55703, or amutant strain thereof possessing substantially all of the identifyingcharacteristics of the microorganism; and

a cellulosic material.

In accordance with another aspect of the present invention, theforegoing and other objects are achieved by:

a composition comprising:

a cellulase produced by a microorganism consisting of ATCC 55702, or amutant strain thereof possessing substantially all of the identifyingcharacteristics of the microorganism; and

a cellulosic material.

In accordance with another aspect of the present invention, theforegoing and other objects are achieved by:

a method of treating a cellulosic material comprising:

contacting the cellulosic material with an amount of a cellulaseproduced by a microorganism consisting of ATCC 55703, or a mutant strainthereof possessing substantially all of the identifying characteristicsof the microorganism.

In accordance with another aspect of the present invention, theforegoing and other objects are achieved by:

a method of treating a cellulosic material comprising: contacting thecellulosic material with an amount of a cellulase produced by amicroorganism consisting of ATCC 55702, or a mutant strain thereofpossessing substantially all of the identifying characteristics of themicroorganism.

In accordance with another aspect of the present invention, theforegoing and other objects are achieved by:

a detergent comprising:

a cellulase produced by a microorganism consisting of ATCC 55703, or amutant strain thereof possessing substantially all of the identifyingcharacteristics of the microorganism; a surfactant; and a builder.

In accordance with another aspect of the present invention, theforegoing and other object are achieved by:

a detergent comprising:

a cellulase produced by a microorganism comprising of ATCC 55702, or amutant stain thereof possessing substantially all of the identifyingcharacteristics of the microorgaism;

a surfactant; and

a builder.

In accordance with another aspect of the present invention, theforegoing and other objects are achieved by:

a bacterial cloning host consisting of ATCC 55702, or a mutant strainthereof possessing substantially all of the identifying characteristicsof said bacterial cloning host.

In accordance with another aspect of the present invention, theforegoing and other objects are achieved by:

an enzyme produced by a microorganism consisting of ATCC 55703, or amutant strain thereof possessing substantially all of the identifyingcharacteristics of said microorganism.

In accordance with another aspect of the present invention, theforegoing and other objects are achieved by:

an enzyme produced by a microorganism consisting of ATCC 55702, or amutant strain thereof possessing substantially all of the identifyingcharacteristics of said microorganism.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a temperature growth curve for the bacteriacorresponding to ATCC 55703 and ATCC 55702, in accordance with oneaspect of the present invention.

FIG. 2 illustrates a pH stability curve for the bacteria correspondingto ATCC 55703 and ATCC 55702, in accordance with one aspect of thepresent invention.

FIG. 3 illustrates a salt tolerance growth curve for the bacteriacorresponding to ATCC 55703 and ATCC 55702, in accordance with oneaspect of the present invention.

FIG. 4 illustrates a comparison of cellulase (CMCase) activity inculture supernatants of the bacterium corresponding to ATCC 55702compared to the bacterium corresponding to ATCC 55703, in accordancewith one aspect of the present invention.

FIG. 5 illustrates an ultraviolet kill curve for a rec- equivalentcellulose degrading bacterium corresponding to ATCC 55702, as comparedto an E. coli rec- and ATCC 55703, in accordance with one aspect of thepresent invention.

DETAILED DESCRIPTION

The importance of microorganisms that are capable of producingcellulases has increased with new demands for industrial processes todegrade or modify cellulosic biomass to commercially importantmaterials. The enzymes produced by those microorganisms are not onlyimportant in conversion of cellulosic waste to commercially useful feedstocks but also to degrade cellulose and hemicellulose to materials thatcan be used in the production of alternative fuels. A wide variety ofanaerobic and aerobic bacterial cellulase producers have been describedincluding the bacterium NCIB 10462. This organism was originally namedPseudomonas fluorescens var. cellulosa. The bacterium has also beencalled Pseudomonas fluorescens subsp. cellulosa in recent publications.This bacterium was isolated from soil in the early 1950's and wasdescribed by Ueda. The bacterium was placed in the National Council forIndustrial Bacteriology (hereinafter referred to as NCIB) repository inScotland, and was given the designation "NCIB 10462." Interest in thestudy of NCIB 10462 has recently increased along with the recent upsurgeof interest in biotechnological conversion of cellulose.

When a sample of NCIB 10462 was recently examined, it was found to havevery few identifying characteristics of Pseudomonas fluorescens var.cellulosa, most noticeably the lack of fluorescence. This led to theconclusion that a new microorganism, which possessed enhanced abilitiesto produce large amounts of celulases and other enzymes, had beendiscovered. The microorganism's original designation has been slightlyaltered to reflect the lack of fluorescence, and will be currentlyreferred to as Pseudomonas cellulosa #28. This designation may be mendedat a later time, once sufficient taxonomic studies have been completed.Peudomonas cellulosa #28 was deposited in the American Type CultureCollection (hereinafter referred to as ATCC) and was given Accession No.55703 (hereinafter referred to as ATCC 55703). ATCC 55703 was thensubjected to intense genetic modification using mutagenesis byN-methyl-N'-nitro-N-Nitrosoguanidine (hereinafter referred to as MNNG)treatment. The protocol for this mutagenesis treatment is fullyexplained in Experiments in Molecular Genetics, Miller, pp 125-129(1972), the entire disclosure of which is incorporated herein byreference. MNNG was utilized because it induces a high frequency ofmutations at doses which result in little killing. The resultingmicroorganism was then designated as Pseudomonas species #142 (orPseudomonas sp. #142). This microorganism was then deposited in the ATCCand was given Accession No. 55702 (hereinafter referred to as ATCC55702).

Due to the new importance of these novel bacteria as candidates forcommercial biomass conversion, the metabolic and physical properties ofATCC 55703 and ATCC 55702 were examined. ATCC 55703 and ATCC 55702 wereexamined by utilizing a wide variety of standard bacteriological methodswhich included assimilation studies, fatty acid analysis, and lipidanalysis. ATCC 55703 and ATCC 55702 were found to grow aerobically on awide variety of solid and soluble cellulosic materials. ATCC 55703 andATCC 55702 grew in complex medium under reducing conditions and weaklyon complex media under full anaerobic conditions. No anaerobic growthwas observed in liquid cellulosic medium. Standard bacteriologicidentification methods and assimilation studies suggested that these twobacteria have few characteristics corresponding to the genus/species P.fluorescens. Assimilation studies suggested only a low probability matchwith the genus Sphingomonas.

Metabolic and physical characterization of ATCC 55703 and ATCC 55702revealed that they were alkalophilic, non-fermentative, gram negative,oxidase positive, motile, cellulose degrading bacteria. The aerobicsubstrate utilization profile of these bacteria were found to have fewcharacteristics consistent with a classification of P. fluorescens witha very low probability match with the genus Sphingomonas. Totalphospholipid analysis did not reveal the presence of any sphingolipidproduced by these bacteria. ATCC 55703 and ATCC 55702 were found to growbest aerobically but also grew well in complex media under reducingconditions. ATCC 55703 and ATCC 55702 grew slowly under full anaerobicconditions on complex media and did not grow anaerobically on cellulosemedia as the sole carbon source. Total fatty acid analysis (MIDI) ofATCC 55703 and ATCC 55702 failed to group these bacteria with any knowngenus or species (very low probability match with the genus Vibrio).Therefore, until conclusive taxonomic data can be gathered, thesebacteria (ATCC 55703 and ATCC 55702) will continue to be designated asbelonging to the Pseudomonas genus.

ATCC 55703 and ATCC 55702 were maintained on solid media consisting ofM9 liquid medium to which 15 grams per liter agarose had been addedalong with 0.1% carboxymethyl cellulose (CMC) as the sole carbon source.ATCC 55703 and ATCC 55702 were then transferred to Trypticase soy agarprior to MIDI analysis. Fatty acid analysis by the MIDI system wasperformed on ATCC 55703 and ATCC 55702 growing anaerobically on 5%sheeps blood agar.

All studies to determine optimal pH and temperature for growth wereperformed in Trypticase soy agar broth. Cellulosic media consisted of M9salt solution as previously described. Agar was added to 15 grams perliter for solid media along with soluble cellulosic component (i.e.,carboxymethyl cellulose (CMC)) to 0.1% (w/v). Cellulosic liquid mediaconsisted of M9 salt solution to which strips of filter paper ornewspaper had been added. Alternatively, Avicell (powder) was added to0.1% (w/v).

All bacteriologic assays and growth procedures were performed usingstandard bacteriology techniques, methods and medias. Non-fermentativeanalysis was performed using an API non-fermentor identification system.MIDI analysis was performed using standard protocols.

Growth studies and non-fermentative analysis indicate that ATCC 55703and ATCC 55702 are small non-fermentative gram negative rod bacteriawith few characteristics that are typically associated with P.fluorescens or even with the genus Pseudomonas. Specifically, Table 1,below, illustrates the similarities and differences between ATCC 55703and ATCC 55702 with respect to P. fluorescens regarding standardbiochemical assays:

                  TABLE 1                                                         ______________________________________                                                                    ATCC 55703 and                                    Test            P. fluorescens                                                                            ATCC 55702                                        ______________________________________                                        Growth on Blood Agar                                                                          +           -                                                 Growth on McConkys Agar                                                                       +           -                                                 Growth in Pseudomonas M10                                                                     +           -                                                 Medium                                                                        Catalase        Strong Positive                                                                           Weak Positive                                     Growth in Thioglycollate                                                                      Aerobic     Anaerobic                                         Fluorescent     +           -                                                 Oxidase         +           +                                                 Tryptophanase   -           -                                                 Arginine dihydrolase                                                                          +           -                                                 Urease          -           -                                                 Esculin         -           +                                                 Gelatinase      +           -                                                 PNGP            -           +                                                 Glucose         +           +                                                 Arabinose       +           +                                                 Mannose         +           -                                                 n-Acetyl-Glucosamine                                                                          +           +                                                 Maltose         -           +                                                 Gluconate       +           -                                                 Caprate         +           -                                                 Adipate         +           -                                                 Malate          +           -                                                 Phenylacetate   ±        -                                                 ______________________________________                                    

Table 2, below, illustrates various morphological and biochemicalcharacteristics of both ATCC 55703 and ATCC 55702:

                  TABLE 2                                                         ______________________________________                                        Gram Morphology     Small gram negative rod                                                       approximately 0.5 μm                                   Motility            Motile at 25 and 37° C.                            Starch Hydrolysis (amylase)                                                                       Positive for starch hydrolysis                            β-galactosidase                                                                              Positive                                                  β-glucosidase  Positive                                                  Hemicellulase       Positive                                                  Xylanase            Positive                                                  Optimal Growth Temperature                                                                        30-37° C.                                          (complex media)                                                               Optimal pH (complex media)                                                                        pH 7.2-8.0                                                Anaerobic growth (37° C.)                                              Blood Agar          Positive                                                  Trypticase Soy Broth                                                                              Positive                                                  CMC Liquid Medium   Negative                                                  CMC Solid Medium    Negative                                                  Avicel Liquid Medium                                                                              Negative                                                  Filter Paper Liquid Medium                                                                        Negative                                                  Aerobic Growth on Cellulosic Media*                                           CMC                 Positive (solid and liquid)                               Avicel              Positive (solid and liquid)                               Filter paper        Positive (solid)                                          Cellobiose          Positive (solid)                                          Newspaper           Positive (liquid)                                         Cellulose acetate   Positive (liquid)                                         Nitrocellulose      Negative (liquid)                                         Polyacrylamide      Negative (liquid)                                         Growth on Other Media                                                         (by ATCC 55702 only)                                                          20% v/v Furfurol    Positive                                                  20% v/v Cinnimyl Alcohol                                                                          Positive                                                  20% v/v Saccharinic Acid                                                                          Positive                                                  ______________________________________                                         *Standing or shake cultures at 30 or 37° C.                       

ATCC 55703 and ATCC 55702 can utilize a wide variety of solid andsoluble cellulosic media as a sole carbon source which is consistentwith previous observations. ATCC 55703 and ATCC 55702 are oxidase andcatalase positive, motile, and aerobic, which are the fewcharacteristics that suggest it might be related to the Pseudomonads.ATCC 55703 and ATCC 55702 only weakly clot a lymulus lysate test whichis interesting since they appear to be 0.5-1 μm gram negative rods usinggram stain. These studies show that ATCC 55703 and ATCC 55702 are notclosely related to the genus Pseudomas, but may have a very lowprobability match to the genus Sphingomonas.

Table 3, below, illustrates additional biochemical characteristics ofATCC 55703:

                  TABLE 3                                                         ______________________________________                                        Indole Production    Negative                                                 N-acetyl glucosaminidase                                                                           Positive                                                 α-Glucosidase  Positive                                                 α-Arabinosidase                                                                              Positive                                                 β-Glucosidase   Positive                                                 α-Fucosidase   Negative                                                 Phosphatase          Negative                                                 α-Galactosidase                                                                              Positive                                                 β-Galactosidase Negative on An-Ident*                                    Indoxyl-acetate hydrolysis                                                                         Positive                                                 Arginine Utilization Negative                                                 Leucine aminopeptidase                                                                             Positive                                                 Proline aminopeptidase                                                                             Positive                                                 Pyroglutamic acid arylamidase                                                                      Negative                                                 Tyrosine aminopeptidase                                                                            Negative                                                 Arginine aminopeptidase                                                                            Positive                                                 Alanine aminopeptidase                                                                             Positive                                                 Histidine aminopeptidase                                                                           Positive (weak)                                          Phenylalanine aminopeptidase                                                                       Positive                                                 Glycine aminopeptidase                                                                             Positive                                                 ______________________________________                                         *Colonies of ATCC 55703 and ATCC 55702 are positive for galactosidase         after incubation with Xgal                                               

With reference to FIG. 1, the growths of ATCC 55703 and ATCC 55702 atvarious temperatures are illustrated. ATCC 55703 and ATCC 55702 have anoperating temperature range of 5-37° C., a preferred temperature rangeof 25°-32° C., and an optimal temperature of 30° C. No growth of ATCC55703 or ATCC 55702 has been observed at 45° C. With respect to thecellulase produced by ATCC 55703 and ATCC 55702, the operatingtemperature range is 5°-70° C. (at pH 7.5). The preferred temperature is55° C. (at pH

With reference to FIG. 2, ATCC 55703 and ATCC 55702 have an operating pHrange of 6.8 to 9.0, a preferred pH range of 7.2 to 8.0, with growthdecreasing after the pH is adjusted below pH 7.0. In complex media, ATCC55703 and ATCC 55702 reduced the medium pH below pH 6.8, whereas inliquid cellulosic medium the pH does not fall more than 0.3 from thestarting pH of 7.2. With respect to the cellulase produced by ATCC 55703and ATCC 55702, the operating pH range is 5-11 (at 55° C.). Thepreferred pH range is 7-8 (at 55° C.).

With reference to FIG. 3, the salt tolerance curve illustrates that ATCC55703 and ATCC 55702 have increased tolerance to twice as much sodiumthan is commonly used in growth medium. With reference to FIG. 3, ATCC55703 and ATCC 55702 have an operating salt range of 8.5-32 mM sodium,with a reference of 8.5 mM sodium. Less dilution of high salt brinesused in paper processing will be required to use ATCC 55703 and ATCC55702 to biodegrade cellulosic waste from the paper industry.

Taxonomic characterization of ATCC 55703 and ATCC 55702 using totalfatty acid analysis failed to group these bacteria with any of thecurrently recognized genera. Since the non-fermentative analysissuggested that ATCC 55703 and ATCC 55702 may be distantly relatedmembers of the genus Sphingomonas, a total lipid analysis was alsoperformed on ATCC 55703 and ATCC 55702. Results indicated that nosphingolipid is produced by ATCC 55703 or ATCC 55702 and the predominantphospholipids are acyl-linked gycerophosphatides.

Since aerobic substrate utilization studies suggested that ATCC 55703and ATCC 55702 might have a very low probability match to the genusSphingomonas, we examined the lipids produced by ATCC 55703 and ATCC55702 with a special interest in the production of sphingolipid. Thelipid profiles of ATCC 55703 and ATCC 55702 are unremarkable. The polarlipid fractions were comprised of ester-linked acyl-glycerophophatides,which would rule out classification of ATCC 55703 or ATCC 55702 in genusSphingomonas.

Total fatty acid profiling has been used to identify anaerobic andaerobic bacteria. Total fatty acid analysis failed to adequately groupATCC 55703 or ATCC 55702 into any known genus. Only a low probabilitymatch with the genus Vibrio was obtained. Total fatty acid analysisfound that a large percentage of the fatty acids produced by ATCC 55703and ATCC 55702 are in the trans configurations which is characteristicof some members of Vibrio. Ribosomal phylogenetic analysis will probablymore dosely define the taxonomic position of these bacteria.

Both ATCC 55703 and ATCC 55702 are capable of degrading a wide varietyof soluble and solid cellulosic materials. Specifically, ATCC 55703 andATCC 55702 have improved characteristics and qualifies for thedegradation of cellulosic waste, degradation of cellulosic fossil fuels(i.e., coal) for alternative fuel production, detergents, drain pipecleaners, septic tank cleaners/additives, textile processing, and paperprocessing. However, ATCC 55702 has demonstrated a far greater abilityto produce large amounts of cellulase than ATCC 55703. FIG. 4 shows thatthe production of extracellular cellulase from ATCC 55702 isapproximately 300 times higher than ATCC 55703. After 24 hours, the zoneof clearing around the well of ATCC 55702 is very large (upper righthand corner) (arrow) compared to ATCC 55703 or eleven other cellulasedegrading bacteria (no zones visible). Table 4, below, illustrates thatATCC 55702 is superior in (1) production of cellulase, (2) degradationof solid cellulose, and (3) conversion of solid cellulase to sugars whencompared to 11 other cellulose degrading bacteria isolated from termitegut. ATCC 55702 is superior in: (1) time to degrade solid cellulosesubstrates; (2) total sugars produced from common cellulosic wastes(i.e., newsprint); and (3) extracellular endocellulase activity.

                  TABLE 4                                                         ______________________________________                                                  Filter Paper                                                                             Extracellular                                                                             Total                                                  Degradation                                                                              Cellulase   Carbohydrate                                 Species   (Days)*    (mm) (Day 5)**                                                                            (μg/ml)****                               ______________________________________                                        ATCC 55702                                                                              3          12          36.5                                         Species 1***                                                                            >7         0           0                                            Species 2 >7         0           0                                            Species 3 >7         0           0                                            Species 4 4          0           18.1                                         Species 5 >7         0           0                                            Species 6 >7         0           0                                            Species 7 >7         0           0                                            Species 8 6          0           0                                            Species 9 >8         0           0                                            Species 10                                                                              >8         0           0                                            Species 11                                                                              6          0           0                                            ______________________________________                                         *First appearance of filter paper structure is degrading                      **Plate diffusion assay zone based on CMC hydrolysis                          ***Strains isolated from termite gut and include Bacillus sp., Pseudomona     sp., and Flavobacterium sp. like                                              ****Total carbohydrate in culture supernatants after 5 days growth on         newspaper                                                                

It has also been found that for ATCC 55702 to grow rapidly on solidcellulose and produce the highest amounts of extracellularendocellulase, it must also demonstrate high levels of β-glucosidase(cellobiase) activity. This enzyme converts cellobiose to glucose. ATCC55702 rapidly produces a colored product from X-GLU (a syntheticmarker/test for β-glucosidase production). Therefore, ATCC 55702 isenhanced not only in the production of extracellular cellulases(endoglucanases), but is also enhanced for β-glucosidase activity.

ATCC 55702 is especially suited to biomass conversion of cellulosicwaste for alternative fuel production. ATCC 55702 grows best andproduces cellulases at temperatures lower than 37° C., a temperaturemost often used to grow most bacteria and fungi. Lower temperaturerequirements means that the yield from biomass conversion by ATCC 55702is more suitable for energy production. The energy required to producethe fuel by growing ATCC 55702 at 20°-24° C. is much less than heatingthe fermentor or bioreactor to 37° C. ATCC 55702 is greatly superior foralternative fuel production as compared to the thermophilic cellulosedegrading bacteria whose growth requires temperatures ranging from45°-65° C.

In accordance with one aspect of the present invention, a simple powdercontaining the cellulase and bacteria can be used as an additive tocellulosic waste (i.e., diapers, tampons, newsprint, etc.). The productwould be embodied as an inexpensive dried or lyophilized powder of thetotal fermentate which contains bacteria and cellulase. No postfermentation ("down stream") processing would be required. The productwould only require fermentation, lyophilization or drying, andpackaging. Summarizing, the powder could consist alternatively of: (1)dried fermentate (cellulase) plus cells (bacteria); (2) dried cells(bacteria); or (3) dried fermentate (cellulase). Additionally, theproduct could be packaged in a liquid format. Thus, the liquid productcould consist alternatively of: (1) fermentate (cellulase); (2) lysedcells (bacteria); or (3) fermentate (cellulase) plus lysed cells(bacteria).

Introduction of new genetic traits into bacteria are inhibited by theenzymes which repair the genetic material (DNA repair andrecombination). Thus, any trait introduced into a bacterium by eitherclassical methods (mutagenesis) or recombinant DNA technologies (geneticengineering) can be destroyed by rearrangement of the genetic materialby the DNA enzymes. In the most common cloning vehicle Escherichia coli,recombinase deficient cloning hosts have been created that lack theserecombinase genes (rec- strains). However, cloning of cellulase genesinto E. coli is not commercially practical since the organism cannotsecrete the cellulase. Further, introduction of cellulase genes isinefficient when compared to bacteria that naturally have the ability todegrade cellulose. Restrictions on the use of recombinant DNA technologyto give a new metabolic capability (i.e., cellulase production) to thoseorganisms that do not have it previously, make commercial use of therecombinant bacteria prohibitive. The ideal solution is to enhance theability of a bacteria that already produces cellulase using standard andrecombinant DNA technologies.

ATCC 55702 would therefore be an improved host for genetic manipulationsusing recombinant DNA techniques, and is less likely to destroy geneticmanipulations using standard mutagenesis techniques. ATCC 55702 isequivalent to Escherichia coli recombinant deficient (rec-). Since rec-cloning hosts cannot repair or recombine DNA, they are very susceptibleto the killing by ultraviolet (UV) irradiation. FIG. 5 illustrates a UVkill curve performed on ATCC 55703, which is UV resistant. Conversely, arec- E. coli cloning host is rapidly killed by slow exposure to UVradiation (FIG. 5). Additionally, the kill curve of ATCC 55702 (a UVsensitive (rec- equivalent) cellulose degrading bacterium) is shown inFIG. 5. Therefore, the ATCC 55702 cloning host will be less able torecombine/repair DNA as efficiently as the wild type bacterium. Thisfeature limits the cloning host from "moving" the genetic elements anddestroying them via DNA repair or combination mechanisms.

Ideal cloning hosts usually require that they be sensitive to two commonantibiotics (tetracycline and ampicillin) that are used to select forrecombinants. ATCC 55703 was resistant to ampicillin and was partiallyresistant to tetracycline. Selection via replicate plating during theprocess of developing the ATCC 55702 rec-strain produced a strain thatwas also highly sensitive to tetracycline and ampicillin. Therefore, inaccordance with one aspect of the present invention, the ATCC 55702cloning host is highly sensitive to these two antibiotics, which isillustrated in Table 5, below:

                  TABLE 5                                                         ______________________________________                                                   10 μg/ml                                                                            50 μg/ml                                                                             100 μg/ml                                                                         200 μg/ml                             Antibiotic conc.    conc.     conc.  conc.                                    ______________________________________                                        Streptomycin                                                                              s*      s         s      s                                        Tetracycline                                                                             r        s         s      s                                        Chloramphenicol                                                                          s        s         s      s                                        Ampicillin r        r         s      s                                        Penicillin G                                                                             r        r         r      r                                        Gentamicin s        s         s      s                                        ______________________________________                                         *s = sensitive, r = resistant                                            

As previously mentioned, ATCC 55703 was subjected to an intensivemutagenesis regimen using MNNG. Resulting strains were selected for: (1)improved soluble cellulase production; (2) rapid degradation of solidcellulose and waste products (filter paper, newsprint and diapers); (3)UV sensitivity; (4) enhanced β-glucosidase activity; (5) tetracyclineand ampicillin sensitivity; and (6) high levels of total carbohydrateproduction from solid cellulose. As a result of replicate platingprocedures, ATCC 55702 was identified.

The present invention has many applications with respect to detergents,drain pipe cleaners, septic tank cleaners/additives, conversion ofcellulosic materials to animal feedstocks, conversion of starches tosugars, increased lignin and hemicellulose removal from cellulosicmaterials, textiles, food processing, enzymes, detergents, paperproducts, bioremediation, specialty chemicals, consumer goods, andrenewable fuels.

DEPOSIT OF MICROORGANISMS

The Applicant, in accordance with the provisions of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure, did deposit samples of Pseudomonascellulosa #28 and Pseudomonas species #142 with the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852,United States of America on Aug. 17, 1995 and was assigned accessionnumbers ATCC 55703 and ATCC 55702, respectively. Both of thesemicroorganism are readily available to the public, in accordance withapplicable rules and regulations.

While there has been shown and described what are present considered thepreferred embodiments of the invention, it will be obvious to thoseskilled in the art that various changes and modifications can be madetherein without departing from the scope of the invention defined by theappended claims.

What is claimed is:
 1. A bacterial strain having all of the identifyingcharacteristics of ATCC
 55702. 2. The strain of claim 1, wherein thestrain is ATCC
 55702. 3. The strain of claim 1, wherein the strain is amutant of ATCC
 55702. 4. A bacterial cloning host which is sensitive toampicillin, tetracycline and UV radiation consisting of ATCC 55702, or amutant strain thereof, possessing all of the identifying characteristicsof said bacterial cloning host.